Fourier transform mass spectrometry

Abstract

This article provides an introduction to Fourier transform-based mass spectrometry. The key performance characteristics of Fourier transform-based mass spectrometry, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of Fourier transform-based mass spectrometry technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for an application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.

Fig. 1.

Fragmentation (MS/MS) spectrum of flavonoid quercetin (m/z 303). Mass deviation of less than 3 ppm for any detected fragment together with the richness of the fragmentation spectra itself enable confirming the elemental composition of the starting compound as well as providing useful hints to its structure. Assignment of the peaks performed using software package Mass Frontier^TM.

Fig. 2.

Effect of mass accuracy on peptide identification, an example of tryptic peptides from a human protein database. A, Graph shows how many peptides would match a search criterion based on the accurate mass of the peptide alone, considering mass deviation of 50, 20, 10, 5, 1, 0.1, and 0.01 ppm, respectively. B, Detail of graph A. Slicing the analyzed mass range into bins with a width corresponding to the respective mass deviation and counting number of peptide hits in each such bin reveals that even with a really low mass deviation one would still encounter many bins with more than 1 peptide in it. C, Histograms for mass bin width of 5, 2, 1 and 0.1 ppm, respectively, where number of peptide matches in a bin is on the x axis and the frequency of an occurrence on the y axis. Courtesy of David Fenyo, Rockefeller University.

Fig. 3.

High resolution results in better mass accuracy. Pesticide Pirimicarb was measured in a mixture of other 115 pesticides and food toxins in a horse feed matrix. Top panel shows a mass spectrum taken at the time of the elution of Pirimicarb acquired at resolving power 15,000 FWHM. The unresolved interferences caused the measured mass of Pirimicarb to be skewed toward a slightly higher value resulting in a mass deviation of 6.5 ppm. The same sample was then acquired at resolution 80,000 FWHM (bottom panel). This resulted in improved accuracy of both mass measurement (0.3 ppm mass deviation) and quantitation. Courtesy of Markus Kellmann, Thermo Fisher Scientific.

Fig. 4.

Illustration of signal complexity encountered in FTMS. A, Four frequencies representing four different masses are considered here: frequency “v” of an intensity 1, frequency “2v” of an intensity 0.5, frequency “5v” of an intensity 1.5, and frequency “8v” of an intensity 0.2. B, Displayed waveforms for the considered intensities and frequencies. C, All four waveforms combined into a signal emulating the time domain signal detected by an FTMS.

Fig. 5.

Phase dependence of the real and imaginary part of Fourier transform solution. A, At initial time phase equal to zero, the real and imaginary part of the complex (real and imaginary) frequency spectrum represents the pure absorption and dispersion mode spectrum, respectively. B, For any other initial time domain phase, the complex (real and imaginary) components represent a linear combination of absorption and dispersion modes, and the resulting peak shapes are asymmetric. Employing a ”magnitude spectrum” removes the initial time domain phase dependence with a penalty of obtaining a spectrum with only half the resolution achievable for that particular data.

Fig. 6.

Example of apodized and nonapodized simulated spectral peak. Whereas apodization improves dynamic range and appearance of FTMS spectra, it impacts negatively on its resolution. Courtesy of R. Malek, Thermo Fisher Scientific.

Fig. 7.

Ion motion within an ICR cell. A, Force affecting a charged particle moving in the homogeneous magnetic field causes the particle to assume circular trajectory. B, As the magnetic field can confine the ions only in the radial direction, no confinement exists in the axial direction along the magnetic field.

Fig. 8.

Techniques applied in FTICR to excite a whole range of m/z simultaneously.

Fig. 9.

Grid cell used to minimize the excitation field perturbations. Graphical rendering of the ICR cell of the LTQ FT Ultra instrument depicting the grids placed inside the cylindrical electrodes over the entire length of the ICR cell. The excitation waveforms are supplied to these grids so that the excitation field extends well past the trapping region. The trapping rings are segmented because the potentials applied to the segment behind the grids have to be 4.6-fold higher than those applied to the grid-free segments in order to establish the same trapping potential.

Fig. 10.

Effect of magnetic field strength on resolution. Resolution (FWHM) achievable for a range of masses for a 1 s transient employing magnetic field strengths of 1 to 40 T is presented. The resolution shown for FTICR is nonapodized. Please note that both axes are logarithmic.

Fig. 11.

Effect of transient duration on resolution. Resolution (FWHM) achievable for a range of masses with magnets of 7 and 21 T and for durations of transients of 1 and 5 s is provided for comparison. The resolution shown for FTICR is nonapodized. Please note that both axes are logarithmic.

Fig. 12.

ECD fragmentation of an intact protein. Charge state 12⁺ of ubiquitin was fragmented using ECD. From the 72 theoretically cleavable peptide bonds within ubiquitin sequence 71 have been cleaved obtaining 147 fragment ions in total. Courtesy of M. Zeller, Thermo Fisher Scientific.

Fig. 13.

Orbitrap mass analyzer. Ions are captured in a quadro-logarithmic electrostatic field (see the equation insert). An outer electrode enclosing a central spindle electrode consists of two halves separated by a dielectric material. The image current of ions moving as concentric rings along the central electrode (oscillations in axial direction denoted as z in the drawing) is picked up by the outer electrode sections.

Fig. 14.

Resolution achievable with FTICR and Orbitrap analyzers. A transient duration of 1 s is considered for FTICR with field strength 9.4 and 21 T, and for two different designs of the Orbitrap analyzer. The resolution shown for FTICR is nonapodized whereas the resolution shown for Orbitrap is apodized. In both types of FTMS the resolution drops with increasing the mass of an analyte. Within experimental settings used in chromatographic applications in proteomics (1 s transient) the Orbitrap system can outperform the FTICR with even very strong magnets for analytes with large m/z.

Fig. 15.

Schematic representation of a hybrid ion trap-Orbitrap mass spectrometer. The main parts of a commercially available hybrid FTMS instrument, the LTQ Orbitrap, are highlighted on the diagram.

Fig. 16.

Spectrum of a monoclonal antibody acquired during an LC/MS analysis with Orbitrap detection. Molecules of immunoglobulin G (approx. 147 kDa) accept a range of different charges during electrospray ionization process, which are then represented as different charge states in the spectrum. The most intense species here carries 55 charges while increasing charge states appear to the left and decreasing to the right thereof. Courtesy of Zhongqi Zhang and Pavel Bondarenko, Amgen.

Fig. 17.

Intact protein analysis with high resolution mass spectrometry. Top panel shows the charge state envelope of myoglobin. A section of the spectrum with a species carrying 15 charges is presented on middle panel, and it is further enlarged to show the individual isotopomers on bottom panel.

Fig. 18.

Phase correction in the Orbitrap analyzer. Diagram showing three different ion signals (frequencies) that can be traced back to the point in time when they have an identical phase. This “time = 0” corresponds to the time the ions leave the C-trap to enter the Orbitrap field.

Fig. 19.

Resolution improvement using phase correction in the Orbitrap analyzer. Charge state 47⁺ of intact yeast enolase (46.64 kDa) was detected in the standard Orbitrap analyzer (760 ms transients). A, The phase corrected spectrum shows baseline isotopic separation. B, The same experiment without the phase correction achieves isotopic separation at FWHM. The phase correction results in 1.6- to 1.7-fold improvement in resolution. Both spectra have been through different apodization.